Clathrin-coated vesicles (ccv) play important roles in sorting plasma membrane proteins into the endocytic pathway and sorting proteins between the trans Golgi network (TGN) and endosomes. These ccv-mediated pathways are fundamental, conserved elements of eukaryotic cells; pathway defects can cause inherited human disorders and are likely to contribute to multigenic diseases such as cancer, heart disease, and Alzheimer's disease. Also, pathogens such as HIV take advantage of these pathways to infect cells and avoid immune surveillance. The overall goal of this project is to understand the molecular basis of selective protein transport by ccv in normal cells to provide a foundation for understanding how defects can lead to disease. Towards this goal ccv-mediated protein transport has been characterized in the yeast Saccharomyces cerevisiae. During the previous funding period a network of three types of clathrin adaptors that function in transport between the TGN and endosomes has been defined, consisting of the AP-1 complex, Gga proteins, and epsin-related proteins. Analysis of these adaptors in yeast has opened unique avenues to address the mechanism of ccv formation at the TGN and endosomes. Additionally, a novel role for ubiquitin binding by an endocytic BAR domain protein, Rvs167p, has been uncovered. This finding provides an opportunity to define functions for ubiquitin binding in endocytosis other than cargo recognition. A combination of genetic, chemical genetic, biochemical, and live cell imaging strategies will be applied to achieve four specific aims. First, the mechanism of clathrin coat assembly at the TGN/endosomes will be characterized in wild-type and mutant yeast strains using time-lapse live cell imaging of endogenously expressed fluorescent adaptors and clathrin. Second, complementary biochemical strategies will be directed at defining roles for particular adaptors in coat assembly, membrane binding and deformation, and cargo selection. Third, approaches in the first two aims will be extended to determine the functions of conserved TGN/endosome accessory factors in ccv formation, and new accessory factors will be identified. Fourth, the mechanism and function of ubiquitin binding by Rvs167p during endocytosis will be determined. Together these studies are expected to provide significant insights into the fundamental process of ccv formation and protein sorting in pathways between the TGN and endosomes, and during endocytosis, thereby helping to establish a foundation for understanding the roles of these processes in human disease. PUBLIC HEALTH RELEVANCE: A fundamental aspect of animal cell structure and function involves protein transport between compartments within the cell. This project will employ yeast as a model eukaryotic cell to address the mechanism of transport mediated by a specific type of transport carrier, clathrin coated vesicles. Insights provided by this project will help to understand how defects in clathrin-mediated transport contribute to disease.